The proposed studies will focus primarily on supra-molecular aspects of biological membranes. Using a combination of freeze substitution, negative staining, SDS-polyacrylamide gel electrophoresis, and immuno-labeling techniques, we will investigate a) the role of flattened vesicles in the cytoplasm of Pelvatia eggs, which can form transient, reversible connections with the plasma membrane, and which seem to function in osmoregulation; b) the preparation of antibodies to the approximately 210 kD peptides of spot demosomes and the localization of these peptides in intact desmosomes; c) the structural changes in the nuclear envelope membranes of Drosophila eggs during the mitotic cycles. An improved propane jet ultra-rapid freezing device (freezing rate -10,000 degrees/sec) will be used to freeze the egg samples without the use of cryoprotectants, and also to achieve the time resolution and structural preservation of the transient membrane phenomena.